Fig 2: Flavones protect ULK1 and cause increased levels of the autophagy marker LC3-II.A MB135-DUX4i myoblasts were either left un-induced or induced with doxycycline for 5 or 24 h. At the indicated times myoblasts were lysed and analyzed by western blotting with antibodies specific to markers of autophagy. B MB135-DUX4i myoblasts were pre-treated with the indicated concentrations of compounds for 3 h, followed by induction of DUX4 expression for 24 h and western blotting analysis as in (A). Uncropped images of all blots are presented in the Supplementary Information. C MB135-DUX4i myoblasts were treated as in (B) and were then analyzed by western blotting with antibodies specific to RIPK1, RIPK3, phospho-RIPK3, and SIRT1.

Fig 3: Phosphorylated RIP3 and MLKL are increased in atezolizumab-treated human hepatocytes (THLE-2 cells) in a dose-dependent manner. (A) The levels of phosphorylated RIP3, RIP3, phosphorylated MLKL, and MLKL expression were evaluated by Western blotting in WCLs of THLE-2 cells after the cells were treated with either indicated concentrations of ATE or left untreated as a control. Arrows indicate oligomerized phosphorylated RIP3 and MLKL, respectively. (B) Knockdown efficiency of PD-L1 expression was examined by Western blotting in WCLs of THLE-2 cells 48 h after transfection of control siRNA and PD-L1 siRNA. Both control siRNA and PD-L1 siRNA-transfected cells were treated with indicated concentrations of atezolizumab for 24 h. (C) The levels of phosphorylated RIP3 and RIP3 expressions were evaluated by Western blotting in the WCLs of THLE-2 cells used in (B). Arrows indicate oligomerized phosphorylated RIP3. (D) Cell viability was examined in THLE-2 cells after the control and PD-L1 siRNA-transfected cells were treated with indicated concentrations of atezolizumab for 24 h. Western blotting panels shown in this figure are representative of at least two or three independent experiments. Black rounds, cubes, triangles, inverted triangles, rhombuses, hollow circles indicate individual sample tested. ** p-value < 0.01, and ns means no significant change.

Fig 4: Schematic illustration how PUFAs induces ferroptotic cell death in the pancreatic cancers. Exogenous PUFAs including LA and αLA taken up into the cells undergo lipid peroxidation leading to ferroptotic cell death. In addition, ROS accumulation from LA and αLA peroxidation may induce phosphorylation of RIP3 and MLKL leading to membrane disruption.

Fig 5: RIPK3 is a target of PRMT5-mediated symmetric arginine dimethylation. GST-enrichment and probing with antibodies against symmetric dimethyl arginine (αSdmArg) to monitor PRMT5-mediated dimethylation of mRIPK1 (A) and mRIPK3 (B). C Effect of PRMT5 inhibitors GSK591 and LLY283 on RIPK3 methylation monitored by GST-enrichment and immunoblotting (inhibitor treatment: last 6 h before cell lysis). D FLAG-tagged wild-type PRMT5 but not the catalytic-deficient PRMT5 mutant (PRMT5mut, R368A) is capable of enhancing RIPK3 arginine methylation. E siRNA mediated depletion of PRMT5 suppresses symmetric dimethylation of mRIPK3 in HEK293T cells. Efficient depletion of PRMT5 is shown by PRMT5 immunoblots and detection of methylated proteins in the total cell lysates F While both RIPK1 and RIPK3 interact with PRMT5, only RIPK3 is a substrate of symmetric arginine dimethylation.